diff --git a/doc/source/whatsnew/v2.1.0.rst b/doc/source/whatsnew/v2.1.0.rst
index 137f2e5c12211..24def10266ab7 100644
--- a/doc/source/whatsnew/v2.1.0.rst
+++ b/doc/source/whatsnew/v2.1.0.rst
@@ -523,6 +523,7 @@ I/O
 - Bug in :func:`read_html`, tail texts were removed together with elements containing ``display:none`` style (:issue:`51629`)
 - Bug in :func:`read_sql` when reading multiple timezone aware columns with the same column name (:issue:`44421`)
 - Bug in :func:`read_xml` stripping whitespace in string data (:issue:`53811`)
+- Bug in :meth:`DataFrame.to_html` where ``colspace`` was incorrectly applied in case of multi index columns (:issue:`53885`)
 - Bug when writing and reading empty Stata dta files where dtype information was lost (:issue:`46240`)
 - Bug where ``bz2`` was treated as a hard requirement (:issue:`53857`)
 
diff --git a/pandas/io/formats/html.py b/pandas/io/formats/html.py
index 32a0cab1fbc41..151bde4e1c4c2 100644
--- a/pandas/io/formats/html.py
+++ b/pandas/io/formats/html.py
@@ -73,10 +73,16 @@ def __init__(
         self.table_id = table_id
         self.render_links = render_links
 
-        self.col_space = {
-            column: f"{value}px" if isinstance(value, int) else value
-            for column, value in self.fmt.col_space.items()
-        }
+        self.col_space = {}
+        is_multi_index = isinstance(self.columns, MultiIndex)
+        for column, value in self.fmt.col_space.items():
+            col_space_value = f"{value}px" if isinstance(value, int) else value
+            self.col_space[column] = col_space_value
+            # GH 53885: Handling case where column is index
+            # Flatten the data in the multi index and add in the map
+            if is_multi_index and isinstance(column, tuple):
+                for column_index in column:
+                    self.col_space[str(column_index)] = col_space_value
 
     def to_string(self) -> str:
         lines = self.render()
diff --git a/pandas/tests/io/formats/test_to_html.py b/pandas/tests/io/formats/test_to_html.py
index 9c128756339ab..bea0eebef2cf6 100644
--- a/pandas/tests/io/formats/test_to_html.py
+++ b/pandas/tests/io/formats/test_to_html.py
@@ -896,3 +896,59 @@ def test_to_html_float_format_object_col(datapath):
     result = df.to_html(float_format=lambda x: f"{x:,.0f}")
     expected = expected_html(datapath, "gh40024_expected_output")
     assert result == expected
+
+
+def test_to_html_multiindex_col_with_colspace():
+    # GH#53885
+    df = DataFrame([[1, 2]])
+    df.columns = MultiIndex.from_tuples([(1, 1), (2, 1)])
+    result = df.to_html(col_space=100)
+    expected = (
+        '<table border="1" class="dataframe">\n'
+        "  <thead>\n"
+        "    <tr>\n"
+        '      <th style="min-width: 100px;"></th>\n'
+        '      <th style="min-width: 100px;">1</th>\n'
+        '      <th style="min-width: 100px;">2</th>\n'
+        "    </tr>\n"
+        "    <tr>\n"
+        '      <th style="min-width: 100px;"></th>\n'
+        '      <th style="min-width: 100px;">1</th>\n'
+        '      <th style="min-width: 100px;">1</th>\n'
+        "    </tr>\n"
+        "  </thead>\n"
+        "  <tbody>\n"
+        "    <tr>\n"
+        "      <th>0</th>\n"
+        "      <td>1</td>\n"
+        "      <td>2</td>\n"
+        "    </tr>\n"
+        "  </tbody>\n"
+        "</table>"
+    )
+    assert result == expected
+
+
+def test_to_html_tuple_col_with_colspace():
+    # GH#53885
+    df = DataFrame({("a", "b"): [1], "b": [2]})
+    result = df.to_html(col_space=100)
+    expected = (
+        '<table border="1" class="dataframe">\n'
+        "  <thead>\n"
+        '    <tr style="text-align: right;">\n'
+        '      <th style="min-width: 100px;"></th>\n'
+        '      <th style="min-width: 100px;">(a, b)</th>\n'
+        '      <th style="min-width: 100px;">b</th>\n'
+        "    </tr>\n"
+        "  </thead>\n"
+        "  <tbody>\n"
+        "    <tr>\n"
+        "      <th>0</th>\n"
+        "      <td>1</td>\n"
+        "      <td>2</td>\n"
+        "    </tr>\n"
+        "  </tbody>\n"
+        "</table>"
+    )
+    assert result == expected