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This study examines substances from plant leaves of Azadirachta indicaand Melia azedarachextracted with methanol (80%) and then successively fractionated with increasing gradient polarity solvents. The methanol extract from the two plant leaves was selected to be incorporated with nanocomposites because of their high antimicrobial activities. HPLC analysis was performed for the determination of total phenolics and flavonoids. LC–ESI–MS positive ion acquisition mode revealed the presence of four main chemical classes: limonoids, triterpenes, fatty acids, and phenolics. Sol–gel processes were adapted to prepare CaO/chitosan composite loaded with the methanol extract of the two plant leaves, acting as innovative antibacterial agents. The fabricated nanocomposites were characterized by XRD, TEM, SEM, and FTIR methods. Minimal bactericidal concentrations (MBC) were established by agar plating using bacteria treated with MIC and at least 2 concentrations double higher than the MIC. Biofilm was cultivated in the presence of sub-MICs of the extracts and the nanocomposites, followed by quantification with the crystal violet assay. The examined chitosan–CaO-based hybrid nanocomposites loaded with the methanol fractions from the leaves of A. indicaand M. azedarachhad an excellent performance as biofilm inhibitors. This indicates their good potential for biomedical applications.