Stable isotope techniques in food web studies often focus on organic carbon in food sources which are subsequently assimilated in the tissue of consumer organisms through diet. The presence of non-dietary carbonates in bulk samples can affect their δ13C values, altering how their results are interpreted. Acidification of samples is a common practice to eliminate any inorganic carbon present prior to analysis. We examined the effects of pre-analysis acidification on two size fractions of sediment organic matter (SOM) from marine and freshwater wetlands and pure muscle tissue of a common freshwater invertebrate (Cherax destructor). The elemental content and isotopic ratios of carbon and nitrogen were compared between paired samples of acidified and control treatments. Our results showed that acidification does not affect the elemental or isotopic values of freshwater SOM. In the marine environment acidification depleted the δ13C and δ15N values of the fine fraction of saltmarsh and δ15N values of mangrove fine SOM. Whilst acidification did not change the elemental content of invertebrate muscle tissue, the δ13C and δ15N values were affected. We recommend to researchers considering using acidification techniques on material prepared for stable isotope analysis that a formal assessment of the effect of acidification on their particular sample type should be undertaken. Further detailed investigation to understand the impact of acidification on elemental and isotopic values of organic matter and muscular tissues is required.