The cytotoxicity of formaldehyde, glutaraldehyde and their binary mixtures was determined using the MTS (tetrazolium salt; Promega) in vitro assay. Cytotoxicity endpoints were investigated in human cells including; pulmonary type II-like epithelial cell lines (A549), hepatoma cell lines (HepG2) and skin fibroblasts. In order to study the cytotoxic effects of airborne formaldehyde, standard atmospheres at concentrations below 10 ppm (12.3 mg/m3) were generated using a dynamic diffusion method. Formaldehyde was bubbled through serum free culture media and cell viability was investigated after treating cells with formaldehyde air samples. HepG2 cells were found to be more sensitive (IC50 = 103.8 ± 23.6 mg/L) to formaldehyde cytotoxicity than both A549 lung derived cells and skin fibroblasts (P < 0.01). For glutaraldehyde, skin fibroblasts were found to be more sensitive (IC50 = 99.9 ± 17.2 mg/L) than A549 cells (p < 0.01). Mixtures of formaldehyde and glutaraldehyde at a specific range of concentrations, approximately 5-20% (w/w) formaldehyde, induced synergistic effects in A549 cells. Exposing of HepG2 cells with formaldehyde air samples (8.75 ppm × 4 h) reduced cell viability to less than 50% (31.6 ± 1.24%). Our findings emphasized the toxic effects of formaldehyde and glutaraldehyde and their potential toxicity interactions should be taken into consideration for occupational risk assessment of health care workers.