The Antarctic continent hosts heterogeneous regions, which are in general exposed to extreme conditions, mainly low temperatures. Thus, microorganisms that thrive in this environment are supposed to harbor a metabolic versatility of great biotechnological interest, which includes the production of cold-adapted enzymes. The present study aimed to investigate chitinase production by bacteria isolated from marine and terrestrial samples from the Maritime Antarctica. For this, a collection of 560 bacteria was evaluated. Seventy-three (13%) isolates, belonging to 13 bacterial genera affiliated to Proteobacteria (6), Actinobacteria (4) and Firmicutes (3), were positive in preliminary assays, and twenty strains were further selected based on individual plate assays at 5 and 15 °C. These strains were identified such as Arthrobacter psychrochitiniphilus, Arthrobacter cryoconite and Curtobacterium luteum, all belonging to the phylum Actinobacteria. The best chitinase producer was A. psychrochitiniphilus 492, which showed 43.39 U/L in 80 h of incubation. After optimization of nine independent variables by Plackett-Burman experimental design, followed by a fractional factorial (25−1) and a central composite design (CCD) 23, the best condition for chitinase production was achieved using yeast extract 1.0 g/L, NH4NO3 2.0 g/L, colloidal chitin 5.0% and initial pH 6.0, which allowed an increase of 7.7-fold in chitinase production as compared with the initial condition without optimization. Results gathered herein reinforce the Antarctic environment as a rich source for chitinase prospecting and open perspectives for further optimization strategies aiming future biotechnological application of such molecules in ecologically-friendly and sustainable processes.