A bacterial strain capable of utilizing a mixture containing 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic (4-HBA) acid was isolated through enrichment from a soil sample. Based on 16SrDNA sequencing, the microorganism was identified as Acinetobacter calcoaceticus. The sequence of biodegradation of the three isomers when provided as a mixture (0.025%, w/v each) was elucidated. The dihydroxylated metabolites formed from the degradation of 2-HBA, 3-HBA and 4-HBA were identified as catechol, gentisate and protocatechuate, respectively, using the cell-free supernatant and cell-free crude extracts. Monooxygenases and dioxygenases that were induced in the cells of Acinetobacter calcoaceticus in response to growth on mixture containing 2-HBA, 3-HBA and 4-HBA could be detected in cell-free extracts. These data revealed the pathways operating in Acinetobacter calcoaceticus for the sequential metabolism of monohydroxybenzoate isomers when presented as a mixture.