The effect of Lactobacillus fermentum in co-inoculation with Saccharomyces cerevisiae UFLA CA11 on the quality of cachaça (sugar cane spirit) was evaluated. The co-inoculation was first evaluated in flask fermentation, and subsequently, yeast and bacteria were co-cultured at approximately 10(5) CFU/mL and 10(8) CFU/mL, respectively, in 4 consecutive batches. L. fermentum did not affect the growth or activity (sugar consumption and fermentation rate) of S. cerevisiae UFLA CA11 during fermentation. The physicochemical analysis revealed a higher concentration (Tukey test) of aldehydes (22.07 mg/100 mL anhydrous alcohol) in cachaça produced by co-inoculation. Analysis of volatile compounds using GC-FID demonstrated that cachaça produced by co-inoculation had higher concentrations of acetaldehyde (25.15 mg/L), ethyl acetate (30.17 mg/L), and 2,3-butanedione (170.39 μ/L), while cachaça produced by UFLA CA11 contained higher concentrations of ethyl lactate (1205.98 μ/L), propionic acid (127.97 μ/L), butyric acid (2335.57 μ/L), and 1-pentanol (469.23 μ/L). The lowest concentration of acetic acid measured by HPLC was found in cachaça obtained with UFLA CA11. The sensory analysis, performed using the Mann-Whitney test, revealed that cachaça produced by co-inoculation differed from that produced by UFLA CA11 in taste and aroma. This study reports on the use of a mixed culture of Saccharomyces cerevisae and Lactobacillus fermentum to produce cachaça and shows the influence of co-inoculation of yeast and bacteria on the quality of this beverage.