diff --git a/newsfragments/6178.fixed.md b/newsfragments/6178.fixed.md
new file mode 100644
index 00000000000..087e6f6f350
--- /dev/null
+++ b/newsfragments/6178.fixed.md
@@ -0,0 +1,3 @@
+Fix build failure when `VIRTUAL_ENV` or `CONDA_PREFIX` is set but the Python interpreter 
+does not exist at the derived path. PyO3 now checks that the interpreter exists and 
+falls back to other detection methods with a warning if it does not.
diff --git a/pyo3-build-config/src/impl_.rs b/pyo3-build-config/src/impl_.rs
index 0a706fcdceb..2c1bd243641 100644
--- a/pyo3-build-config/src/impl_.rs
+++ b/pyo3-build-config/src/impl_.rs
@@ -2471,8 +2471,32 @@ fn get_env_interpreter() -> Option<PathBuf> {
     match (env_var("VIRTUAL_ENV"), env_var("CONDA_PREFIX")) {
         // Use cfg rather than CARGO_CFG_TARGET_OS because this affects where files are located on the
         // build host
-        (Some(dir), None) => Some(venv_interpreter(&dir, cfg!(windows))),
-        (None, Some(dir)) => Some(conda_env_interpreter(&dir, cfg!(windows))),
+        (Some(dir), None) => {
+            let path = venv_interpreter(&dir, cfg!(windows));
+            if path.exists() {
+                Some(path)
+            } else {
+                warn!(
+                    "VIRTUAL_ENV is set to `{}` but no Python interpreter found at `{}`; ignoring",
+                    dir.to_string_lossy(),
+                    path.display()
+                );
+                None
+            }
+        }
+        (None, Some(dir)) => {
+            let path = conda_env_interpreter(&dir, cfg!(windows));
+            if path.exists() {
+                Some(path)
+            } else {
+                warn!(
+                    "CONDA_PREFIX is set to `{}` but no Python interpreter found at `{}`; ignoring",
+                    dir.to_string_lossy(),
+                    path.display()
+                );
+                None
+            }
+        }
         (Some(_), Some(_)) => {
             warn!(
                 "Both VIRTUAL_ENV and CONDA_PREFIX are set. PyO3 will ignore both of these for \