Thailand is one of the leading exporting countries of rambutan and rambutan peels are considered as a biological waste. In this study, rambutan (Nephelium lappaceum L. cv. Rong Rian) peel extracts (RPE) obtained by water extraction were analyzed for their phytochemical composition, antibacterial and antioxidant activities, and cytotoxicity. The bioactive compounds in RPE identified by GC-MS were mome inositol (35.99 mg/g), catechol (29.37 mg/g), 5-hydroxymethylfurfural (5.69 mg/g), 2-pentenal, (E)-(5.22 mg/g), acetic acid (3.69 mg/g), 1,2,3-propanetriol (3.67 mg/g), 2-furan-carboxaldehyde (2.66 mg/g), and other compounds. FT-IR analysis confirmed the presence of alcohol and phenol in the extract. Antibacterial activities of RPE against food pathogenic and spoilage bacteria showed that RPE could inhibited Bacillus subtilis, B. cereus, Staphylococcus aureus, Vibrio cholerae, V. parahaemolyticus, Pseudomonas aeruginosa, and P. fluorescens, with MIC values ranging between 1024 and 8192 µg/mL. The extract also showed antioxidant properties, as determined by DPPH and ABTS assays. The cytotoxicity analysis after 72 h of treatment showed the IC50 values at 194.97 ± 4.87, 205.92 ± 2.55, and 94.11 ± 1.33 µg/mL for L929, Vero, and MCF-7 cell lines, respectively. Therefore, this study provided a basis of knowledge of rambutan peels as an excellent source of natural bioactive compounds for various applications.