The aim of this study was to determine the ecotoxicological and cytotoxic effects of monoanthraquinone dyes, i.e., Alizarin Blue Black B (ABBB), Acid Blue 129 (AB129), and Remazol Brilliant Blue R (RBBR), using Microbial Assay for Risk Assessment (MARA) and human hepatocellular carcinoma (HepG2) cells. Moreover, the study evaluated the (anti)estrogenic and (anti)androgenic properties of these dyes using Saccharomyces cerevisiae (YES/YAS assays) as a model organism. All dyes showed low (RBBR and ABBB) or slight toxicity (AB129), with average minimum toxic concentrations (MTC) of 151 and 184 mg/L, and 124 mg/L, respectively. Among the tested microorganisms, Brevundimonas diminuta was the most sensitive to all dyes. These dyes demonstrated estrogenic activity at concentrations ranging from 3 to 100 µM, acting as hERα receptor agonists. Both ABBB and RBBR were characterized by agonistic properties towards the hAR receptor at specific concentration (10–100 and 30–100 µM, respectively). ABBB increased mRNA expression of CYP1A1, CYP1A2 and CYP2B6 genes in HepG2 cells along with corresponding cytochrome activities (EROD, MROD and PROD assays). AB129 elevated CYP1A1 and CYP1A2 gene expression but increased only EROD activity. RBBR upregulated CYP1A2 and CYP2B6 gene expression, but did not affect the activity of any measured cytochrome assays.