Structure elucidation of carotenoids pigments present in Rhodococcus sp. and Gordonia sp. was performed with Fourier-transformed infrared spectrometry (FT-IR), high performance liquid chromatography (HPLC), ultravioletvisible spectrometry (UV-Vis), electrospray ion-mass spectrometry (ESI-MS) and proton and carbon nuclear magnetic resonance (1H and 13C NMR). A HPLC carotenoid quantification method was developed, where antioxidant activity with 2,2-diphenyl-1-picrylhidrazyl (DPPH) radical was performed to calculate the inhibitory concentration 50 (IC50) of each carotenoid mixture; contribution of each carotenoid present in mixtures and possible interactions were determined. Rhodococcus sp. and Gordonia sp. presented two and three carotenoids respectively; analytical techniques allowed to suggest that carotenoids present in these strains might have structures similar to the reduced form of 4-oxo retinaldehyde, 8’-apoastaxanthinal and astaxanthin dirhamnoside. Rhodococcus sp., Gordonia sp. and β-carotene presented an IC50 of 1.07, 0.09 y 19.49 μg/mL respectively. Possible interaction in Gordonia sp. between the reduced form of 4-oxoretinaldehyde like carotenoid and astaxanthin dirhamnoside like carotenoid was assessed.