Surfactants such as alkylphenol polyethoxylates (APEOs) and nonylphenol ethoxylates (NPEOs) are commonly used worldwide, but the majority of these compounds, together with their metabolites, have been reported to induce severe biological toxicity. Here, we evaluated for the first time the cytotoxicity, genotoxicity and mitochondrial damage in Chinese hamster ovary (CHO-K1) cells caused by a novel non-ionic surfactant, vanillin ethoxylates (VAEOs), an alternative to APEOs. In parallel, the same in vitro bioassays were conducted on NPEOs along with their metabolic byproducts 4-nonylphenol (4-NP) and vanillin. The results showed that the cytotoxic potency order was NPEOs > 4-NP > VAEOs > vanillin using CCK-8 assays. Also, 4-NP showed potential direct DNA damage in SOS/umu tests, whereas NPEOs, VAEOs and vanillin showed no positive result with and without S9 addition. In addition, none of the test compounds showed obvious genotoxic effects with low olive tail moment value using comet assays. However, all test compounds were shown to cause mitochondrial impairment by increasing mitochondrial mass and decreasing mitochondrial membrane potential in a concentration-dependent manner. And further analysis of reactive oxygen species (ROS) and mitochondrial superoxide (MNSOD) measurement showed that mitochondrial impairment was induced by oxidative stress with intracellular ROS and MNSOD overproduction. It's worth noting that VAEOs and vanillin cause relative lower cytotoxic, genotoxic and mitochondrial damage effects than NPEOs and 4-NP, indicating that VAEOs have the potential to substitute NPEOs as suitable surfactants. Take together, this study elucidates the toxicity profiles of VAEOs and NPEOs relatively comprehensively, and further toxicity analyses are suggested in the population, community and ecosystem.