Operating room personnel have the potential to be exposed to surgical smoke, the by-product of using electrocautery or laser surgical device, on a daily basis. Surgical smoke is made up of both biological by-products and chemical pollutants that have been shown to cause eye, skin and pulmonary irritation. In this study, surgical smoke was collected in real time in cell culture media by using an electrocautery surgical device to cut and coagulate human breast tissues. Airborne particle number concentration and particle distribution were determined by direct reading instruments. Airborne concentration of selected volatile organic compounds (VOCs) were determined by evacuated canisters. Head space analysis was conducted to quantify dissolved VOCs in cell culture medium. Human small airway epithelial cells (SAEC) and RAW 264.7 mouse macrophages (RAW) were exposed to surgical smoke in culture media for 24 h and then assayed for cell viability, lactate dehydrogenase (LDH) and superoxide production. Our results demonstrated that surgical smoke-generated from human breast tissues induced cytotoxicity and LDH increases in both the SAEC and RAW. However, surgical smoke did not induce superoxide production in the SAEC or RAW. These data suggest that the surgical smoke is cytotoxic in vitro and support the previously published data that the surgical smoke may be an occupational hazard to healthcare workers.