Plastic has become a ubiquitous material in aquatic ecosystems. Its mass production has also led to the mass formation of plastic waste, which through various processes in nature becomes microplastic (MP) waste and often ends up in aquatic habitats. Due to its proven toxic effects on living things, MP is considered a global contaminant in freshwater and marine ecosystems, especially regarding the possible transportation through trophic levels, all the way to humans. In water, fish are constantly exposed to the risk of ingestion and accumulation of MP particles in their organism. They can serve as good bioindicators, and previous research indicates a high rate of finding MP particles in the digestive system of fish. Development of methods for isolation and accurate quantification of MP in marine and freshwater habitats is increasingly important. For monitoring of the spatial distribution and temporal trends of marine MP debris, development and implementation of standardized protocols is highly important, in order to efficiently and accurately determine the quantity of MP in different habitats. In this work, the influence of digestion by four different methodologies on isolation and determination of MP particles from the intestine of fish from freshwater and marine ecosystems of the Republic of Croatia were examined. The obtained results indicate that combination of nitric acid, HNO3, and hydrofluoric acid, HF represents the most appropriate methodology for MT isolation from fish intestine, with minimum influence on its structure. The particles of MP were identified in the gut content of all analysed fish, therefore confirming its presence and uptake in aquatic organisms.