ABSTRACT It remains a challenge to expand the hydrophobic application of cis‐1,4‐polyisoprene latex (IRL). Herein, we developed a novel one‐pot strategy to prepare hydrophobic composite films with hexadecyltrimethoxysilane (C16AS) modified diatomite (org‐DE) during the IRL preparation process, achieving composite latex preparation and film functionalization simultaneously. Org‐DE exhibits superhydrophobicity with a water contact angle (WCA) of 156°. A stable org‐DE/IRL composite latex with excellent film‐forming properties was successfully prepared. The characteristics of both the composite latex and its film were evaluated. Org‐DE achieved uniform dispersion in IRL films, demonstrating remarkable film‐forming ability. Compared to the IRL film, all org‐DE(1‐4)/IRL composite films exhibited increased strength, with the composite film of org‐DE(2)/IRL showing a 14.5% increase. As the quantity of the org‐DE rose from 1 to 4 phr, WCA values of the composite films increased from 90° to 112°, which is highly beneficial for improving their dust and stain resistance. Furthermore, the composite films' hydrophobicity can be controlled and adjusted according to specific requirements. The films can be used for medical gloves, hydrophobic rubber gloves, latex clothing, coating materials, rubber boots, or monolayer and multilayer products, thereby broadening the application of IRL.