Ethnopharmacological relevance: There are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms. Materials and methods: Quantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy. Results: It was observed the presence of AZA in the extract (0.1470.02 mg/L). Testing Neem EE subinhibitory concentrations, a significant biofilm adherence inhibition from 62.5 mg/mL for a sensitive S. aureus and 125 mg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000 mg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50 mg/mL at 72 h of incubation, while 300 mg/mL at 24 h of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96 h of incubation with 100 mg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300 mg/mL (male) and 100 mg/mL (female) of the extract showed severe morphological changes after 24 h of treatment. Conclusions: Neem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.