Background: Fruiting bodies of two oyster mushroom (Pleurotus ostreatus and Pleurotus florida) species and their respective substrate samples cultivated and collected from the Mushroom Research, Production and Training Laboratory of the Haramaya University, Ethiopia, were analyzed for essential and non-essential metals, viz., Cu, Fe, Zn, Mn, Cd and Pb contents by flame atomic absorption spectrometry after digestion employing dry, wet and microwave digestion techniques. Concentrations of the analyzed metals were expressed on a dry weight basis (mg/kg). Results: The mean metal concentration (mg/kg in dry weight) in the mushroom and respective substrate samples, respectively; for Pleurotus ostreatus were: Cu (51.19, 48.97), Fe (220.87, 299.51), Zn (89.68, 62.55) and Mn (47.55, 58.32). The mean metal concentration (mg/kg in dry weight) in mushroom and the respective substrate samples, respectively, for Pleurotus florida were: Cu (53.56, 51.87), Fe (243.92, 265.23), Zn (95.26, 64.25) and Mn (41.29, 62.55). However, Cd and Pb was not detected in both mushroom and substrate samples. The bio-accumulation factor of the heavy metals was calculated and a highest accumulation of Cu and Zn from substrate was observed for all the analyzed mushrooms samples. The observed results were also compared with the reported results.