Mycobacteria form cellulose-containing biofilms and exhibit drug resistance at infection sites. However, the function of mycobacterial biofilms in host defense remains unclear. Herein, we demonstrate that reductive stress-induced mycobacterial biofilms evade macrophage capture and protect bacilli from nitric oxide stress. We first determined the optimal conditions for biofilm formation by mycobacteria. Using green fluorescent protein (GFP)-labeled mycobacteria, we then examined the protective effect of biofilms on the capture of bacilli by macrophages and found that macrophage capture was inhibited in the presence of biofilms. Furthermore, we constructed GFP-expressing mycobacteria that respond to acidic pH and nitric oxide stress, both of which are bactericidal factors. The results showed that mycobacterial biofilms protect bacilli from nitric oxide-mediated stress, but not from acidification. Finally, the removal of biofilms by cellulase enhanced the capture of mycobacteria by macrophages and the exposure of mycobacteria to nitric oxide. These findings highlight the protective roles of mycobacterial biofilms in innate immunity, particularly against macrophage capture and nitric oxide-induced stress.