Northern Andean highland lakes play a key role in supporting agriculture and local livelihoods, but they are increasingly affected by anthropogenic pressures such as agricultural expansion and urbanization. These pressures can alter water quality and ecosystem functioning, yet their effects on microbial communities remain poorly understood. Here, we analyze bacterial community structure and predicted functional potential across four freshwater lakes in the Eastern Cordillera of Colombia, representing contrasting levels of environmental preservation. Using 16S rRNA gene sequencing, we characterized microbial diversity and community composition in two lakes subject to higher anthropogenic influence (Fúquene and Tota) and two relatively well-preserved systems (Calderona and Colorado). Lakes with lower preservation status exhibited higher microbial richness and diversity, likely associated with increased nutrient inputs and environmental heterogeneity. In contrast, more preserved lakes showed lower diversity but more stable community composition. Spatial heterogeneity also played a key role, with larger systems displaying greater horizontal variability linked to tributary inflows and extensive littoral zones, as well as vertical stratification driven by gradients in light, temperature, oxygen, and nutrients. Co-occurrence patterns among dominant genera suggest environmentally structured microbial associations across lakes. Inferred functional profiles indicate differences in predicted metabolic potential among preservation statuses, particularly in pathways related to carbohydrate metabolism and xenobiotic degradation. However, these results represent inferred functional capacity based on taxonomic composition rather than direct measurements of microbial activity. Overall, our findings indicate that environmental preservation status and spatial heterogeneity shape both the composition and predicted metabolic potential of bacterial communities in Andean freshwater ecosystems. These results provide a baseline for understanding microbial responses to environmental change and highlight the need for future studies integrating metagenomic and experimental approaches to validate ecosystem processes.