BACKGROUND: o-Aminophenol derivatives are of particular interest for their di-verse biological activities and potential therapeutic applications. Such as, antioxidant, an-tibacterial, and cytotoxic activities. OBJECTIVE: This study aimed to design and synthesize a series of novel o-aminophenol de-rivatives through an efficient multi-step process, characterize them using modern spectro-scopic techniques, and evaluate their antimicrobial, antioxidant, and cytotoxic activities. METHODS: A series of novel derivatives of o-aminophenol have been successfully synthe-sized with very high efficiency through a simple six-step process using readily available chemicals and straightforward reactions. The structures of all products were accurately de-termined using modern spectroscopic methods such as 1D and 2D NMR, as well as IR, MS spectroscopy, and X-ray methods. The antimicrobial activities of eight o-nitrophenol derivatives were assessed against Gram (-) and Gram (+) bacteria as well as fungi. In comparison, antioxidant activities were tested for two o-nitrophenol and 11 o-aminophenol derivatives using SC50 and EC50 assays. Cytotoxicity was evaluated on KB, HepG2, A549, and MCF7 cancer cell lines. RESULTS: Six synthesized compounds 5b, 5c, 5g, 6b, 6c, 6g exhibited unusual doublet sig-nals in the H8 region of the 1H NMR spectrum, attributed to atropisomer formation. Eight o-nitrophenol derivatives demonstrated weak antimicrobial activity, with MIC values ranging from 100 to 200 μg/mL. Compound 5g showed activity against all tested bacterial and fungal strains. In antioxidant testing, eight o-aminophenol derivatives 6a, 6b, 6c, 6e, 6f, 6h, 6i, and 12b displayed excellent activity, with SC50 values between 18.95 and 34.26 μg/mL, approaching ascorbic acid's SC50 value of 12.60 μg/mL. Three derivatives 6d, 6g, and 12a showed superior antioxidant activity with EC50 values between 4.00 and 11.25 μg/mL, surpassing quercetin's standard of 9.8 μg/mL. Cytotoxicity assays revealed that o-aminophenol derivatives 6b, 6c, 6f, 6i, and 12b exhibited moderate inhibitory effects on KB cell lines with IC50 values from 32 to 74.94 μg/mL. Compound 6i demonstrated mod-erate cytotoxic activity against HepG2, A549, and MCF7 cell lines, with IC50 values of 29.46, 71.29, and 80.02 μg/mL, respectively. CONCLUSION: Design, synthesis, antimicrobial activity, DPPH Radical Scavenging, Cyto-toxic activity, Evaluation of H8 signal anomalies in certain compounds, and Single crystal X-ray diffraction analysis.