Probiotics and co-culture of shrimp and tilapia are two strategies to improve yield and ecoefficiency of both species. However, only few studies have analyzed the combination of these two strategies. This study analyzes the effect of a mixture of potential probiotics supplied in the diet at different frequencies in a system of shrimp (10/m2) and tilapia (5/m2) in a trial lasting 84 days. The probiotics mixture was composed of four lactic acid bacteria and one yeast strain that were supplied either daily, every 5 days, or every 10 days in the diet and compared with a control without any supply of probiotics. At the end of the experiment, blood chemistry and hematology of shrimp and tilapia were analyzed as an index of physiological condition. Water quality did not differ between treatments. The final weight, feed conversion ratio, and yield of shrimp were significantly higher with daily supply of probiotics compared to shrimp that did not receive probiotics supply, with intermediate values for treatments with probiotics supply every 5 or 10 days. However, level of supplementation every 5 days could be considered as optimal because no significant differences with daily supply were observed for any variable, whereas the supply each 10 days resulted in a significantly lower yield. Significantly higher final weight, feed conversion ratio, and yield of tilapia occurred with daily supplements. Blood cholesterol in tilapia significantly decreased with increasing frequency of probiotics supplementation. These results indicate that probiotics supplements for shrimp and tilapia cultivated simultaneously improved yields and physiological condition.