During tequila production from agave, wastewaters are produced, such as dark-colored vinasse. To add value to this vinasse, microalgae-yeast biomass was produced on vinasse diluted with tequila process water (first rinsing water of agave syrup production). In batch experiments, a vinasse concentration of 10 %v/v resulted in the highest biomass productivity, pH and microalgae growth compared to 20 and 30 %v/v. To ease harvesting, microalgae-yeast flocs (MaY-flocs) were developed in a sequencing batch reactor (SBR). A MaY-floc SBR was run with diluted vinasse (10 %v/v) enriched to 76 mg N-TA L, resulting in a doubled biomass productivity (49.5 ± 8.3 mg VSS L day) of MaY-flocs compared to the best batch reactor performance. Based on response surface experiments, enrichment to 150 mg N-TA L and 5.9 %v/v vinasse are recommended. The MaY-floc SBR system is a promising, novel technology to treat tequila wastewaters while producing settleable MaY-floc biomass of interest to aquaculture.