Increasing introduction of antibiotic residues from humans and animal farming into the environment impacts the functioning of natural ecosystems and significantly contributes to the propagation of antibiotic resistance. Microbial degradation is the major sink of antibiotics in soil but the identification of in situ degrading populations is challenging. Here, we investigated sulfamethoxazole-degrading bacteria in soil microcosms by culture-independent DNA and protein stable isotope probing. 0.5% of the carbon from ¹³C₆-labeled sulfamethoxazole amended to soil microcosms was transformed to ¹³CO₂ demonstrating partial mineralization of the antibiotic. DNA stable isotope probing revealed incorporation of ¹³C from ¹³C₆-labeled sulfamethoxazole into Actinobacteria and among them into the families Intrasporangiaceae, Nocardioidaceae, and Gaiellaceae and the order Solirubrobacterales. Protein stable isotope probing demonstrated the incorporation of ¹³C from ¹³C₆-labeled sulfamethoxazole into proteins of bacteria of the families Intrasporangiaceae, Nocardioidaceae and the order Solirubrobacterales, which is consistent with the results of DNA stable isotope probing. The ¹³C abundance of 60 to 80% in several taxonomically relevant proteins indicated that Intrasporangiaceae directly acquired carbon from ¹³C₆-labeled sulfamethoxazole. The results highlight the crucial role of yet-uncultivated indigenous bacteria for antibiotics degradation, and the potential of cultivation-independent stable isotope based molecular approaches to elucidate the structure of antibiotic-degrading populations in complex microbial communities under natural conditions.