Objective To investigate Acacia honey from different altitudes regarding total phenols and flavonoids, laser-induced fluorescence (LIF) spectra and anticancer activity against human cancer cell lines. Methods Anticancer activity was investigated using sulforhodamine B cytotoxicity assays in the following human cancer cell lines: HCT116 (colon); MCF7 (breast), and HepG2 (liver). Total phenols and flavonoids were measured using spectrophotometric methods and LIF was used to differentiate between low and high-altitude honey. Results The LIF spectra differed between low and high-altitude Acacia honey. High altitude Acacia honey was characterized by significantly lower total phenol content (81.47 ± 1.25 mg gallic acid equivalent [GAE]/100 g) and increased total flavonoids (10.63 ± 0.53 mg quercetin equivalent [QE]/100 g) versus low altitude Acacia honey (91.33 ± 0.96 mg GAE/100 g and 8.78 ± 0.23 mg QE/100 g, respectively). Low altitude Acacia honey displayed increased IC50 values against HCT116 and MCF7 cells (264.17 ± 10.5 and 482.65 ± 20.3 µg/ml, respectively) versus high altitude Acacia honey (117.99 ± 12.7 and 189.82 ± 15.8 µg/ml, respectively). Conclusions High altitude Acacia honey had significantly more effective anticancer activity against HCT116 and MCF7 cells compared with low altitude honey.