The treatment of nitrogen-deficient agriculture wastewater, arising from the vegetable and fruit processing, is a significant problem that limits the efficiency of its biological treatment. This study evaluates the effectiveness of the symbiotic co-culture of Azospirillum brasilense and Scenedesmus sp., under two nitrogen levels (8.23 mg L−1 and 41.17 mg L−1) and mixing systems (aeration and magnetic stirring), aiming to simultaneously use the N-deficient media for their growth while producing biomass for biofuels. Microalgae growth and biomass composition, in terms of protein, carbohydrate and fatty acid contents, were evaluated at the end of the exponential growth phase (15 days after inoculation). Results show that the symbiotic co-culture of microalgae-bacteria can be effectively performed on nitrogen-deficient media and has the potential to enhance microalgae colony size and the fatty acid content of biomass for biofuels. The highest biomass concentration (103 ± 2 mg·L−1) was obtained under aeration, with low nitrogen concentration, in the presence of A. brasilense. In particular, aeration contributed to, on average, a higher fatty acid content (48 ± 7% dry weight (DW)) and higher colony size (164 ± 21 µm2) than mechanical stirring (with 39 ± 2% DW and 134 ± 21 µm2, respectively) because aeration contribute to better mass transfer of gases in the culture. Also, co-culturing contributed in average, to higher colony size (155 ± 21 µm2) than without A. brasilense (143 ± 21 µm2). Moreover, using nitrogen deficient wastewater as the culture media can contribute to decrease nitrogen and energy inputs. Additionally, A. brasilense is approved and already extensively used in agriculture and wastewater treatment, without known environmental or health issues, simplifying the biomass processing for the desired application.