Biomonitoring microplastics in freshwater ecosystems has been insufficient in comparison with its practice in marine environments. It is an important first step to understand microplastic uptake in organisms when assessing risk in natural freshwater habitats. We conducted microplastic biomonitoring within the Greater Melbourne Area; where the microplastic baseline pollution in freshwater organisms was largely unknown. A common noxious fish species, Gambusia holbrooki, was targeted. Individuals (n = 180) from nine wetlands were analyzed. Uptake pathway, size, weight and gender were examined in relation to microplastic uptake in the body (presumed uptake via gut) and head (presumed uptake via gills). On average, 19.4% of fish had microplastics present in their bodies with an abundance of 0.6 items per individual (items/ind) and 7.2% of fish had microplastics in their heads with an abundance of 0.1 items/ind. Polyester was the dominant plastic type and fibers were the most common shape. The amount of microplastics in Gambusia holbrooki in current study is relatively low in a global comparison. The bodies of fish contained more microplastics on average than heads, and the size of microplastics detected in heads were smaller than those found in bodies. Microplastic uptake was directly proportional to size and weight. Furthermore, female individuals showed a tendency to ingest more microplastics than males. Laboratory experiments under controlled conditions are suggested to further explore such relationships. Our findings are important to understanding the potential ecological risks posed by microplastics to organisms in freshwater environments and provide suitable methodologies to conduct biomonitoring in future investigations.