Abstract: The aim of this study was to characterize chitinase-producing bacteria isolated from environmental samples and to investigate conditions affecting chitinase production by these bacteria. Ninety-eight isolates recovered from 20 soil samples were screened for chitinase production. Eighteen isolates showed chitinolytic activity, among which isolate U2 was selected for further study based on dinitrosalisylic acid assay results. The isolate U2 was identified as Massilia timonae through phenotypic characterization and 16S rDNA sequencing and the optimal conditions for chitinase production were determined to be 25-30°C, initial pH 6.0-6.5 and chitin concentration of 1% (w/v). The maximum chitinolytic activity was achieved after 36 h of incubation. The addition of different nitrogen sources to the production medium had no significant effect on chitinase production. Among various carbon sources tested, N-acetylglucosamine (GlcNAc), fructose, lactose, maltose and glucose showed modest inhibitory effect while arabinose did not affect enzyme production by M. timonae isolate U2. The addition of Triton X100 increased chitinase production by 12.4%. The enzyme was reasonably stable in the pH range 5-7 and at temperatures up to 50°C. These results indicate that M. timonae is capable of producing chitinase in relatively simple media containing colloidal chitin as the sole carbon and nitrogen source.