There is lack of data on the prevalence of latex allergy in the health care setting in Iran. This study was performed to determine the prevalence of type I latex allergy and type IV allergy to latex and rubber additives among the operating room staff with glove-related symptoms in 13 general hospitals in Tehran. Skin-prick tests with commercial latex extract, patch tests with latex and 25 rubber additive series, and total and latex-specific IgE detection were performed on the operating room staff who reported latex glove-related symptoms. Five hundred twelve self-administered questionnaires (100%) were completed by all operating room staff and latex glove-related symptoms were reported by 59 (11.5%) employees. Among all symptomatic operating room staff tested, the prevalence of type I latex allergy was 30.5% and the prevalence rates of type IV allergy to latex and rubber additives were 16.7 and 14.6%, respectively. The most positive patch test result with rubber additives was related to tetramethylthiuram monosulfide (38.5%). The risk factors for type I latex allergy were female sex (p = 0.009) and positive patch test with rubber additives (p = 0.012). Subjects who had positive patch test with latex were significantly more likely to have positive patch test with rubber additives (p < 0.0001). Our results showed a high prevalence of type I latex allergy and type IV allergy to latex and rubber additives. Based on this study, we recommend eliminating powdered latex gloves from the operating rooms of the 13 studied general hospitals and support the substitution of powder-free latex gloves.