Nanoplastics (NPs) in aquatic ecosystems have been increasingly detected worldwide and drawn substantial concern due to their small size and hydrophobicity which may pose a threat to aquatic life. In natural aquatic environments, NPs can interact with natural organic matter, like humic acid (HA), by which their surface properties and chemical behaviour are altered. Yet, whether these changes influence the bioavailability and toxicity of NPs to aquatic organisms remains largely unknown. Thus, this study investigated how HA influences the individual and combined toxicity of polystyrene NPs on freshwater microalgae Chlorella vulgaris. NPs with and without surface functionalization (PSNPs and NH₂-PSNPs) were used. Microalgae were exposed to different concentrations of individual PSNPs and NH₂-PSNPs (1-50 mg/L), individual HA (1-10 mg/L), and a combination of HA (1-10 mg/L) with both types of NPs (25 mg/L) for 96 hours. The results showed that in the presence of HA, PSNPs had no effect on the hydrophobicity, heteroaggregation, and co-sedimentation of microalgae, but NH₂-PSNPs exposure reduced all these variables, which is likely driven by the formation of a coating on the surface of NH₂-PSNPs with HA, thereby hindering their transport and internalization in microalgae. However, this reduction was not observed in PSNP-exposed microalgae, due to differences in interaction pathways. When microalgae were exposed to both types of NPs alone, damage to their morphology was observed through SEM and FM due to increased reactive oxygen species production and lipid peroxidation. However, HA addition reduced morphological damage in microalgae exposed NP, regardless of the type and concentration of NPs. The major difference in how different types of NPs interact with HA was the change in hydrophobicity, which alters their toxicity behaviour on microalgae. In conclusion, this study highlights the potential role of HA in modulating the toxicity of NPs on microalgae. The surface properties of NPs also play a significant role in how they interact with HA and affect their toxicity. The results suggest that incorporating natural organic matter into the evaluation of NPs' ecological risks in aquatic ecosystems is crucial. Overall, this study provides insights into the complex interplay between NPs and natural organic matter in aquatic environments, which is essential for developing effective strategies to mitigate their potential environmental impacts.