The present investigation aimed to study the utilisation of combined dairy manure-food waste digestate as a substrate (experimental mushroom substrate—EMS) for Pleurotus djamor (strain 2708, Mycelia) cultivation. Considering the lack of scientific information about the influence of elements concentration in growing substrates on the bioaccumulation of elements in cultivated mushrooms and their residual concentrations in substrates left after cultivation (spent mushroom substrate—SMS), a multi-elemental analysis of 38 elements was carried out. In the study, inductively coupled plasma optical emission spectrometry (ICP OES) was used for elemental analysis. The P. djamor cultivated on EMS resulted in a yield of 196.50 g/bag, achieving a biological efficiency (BE) of 39.90%. High variability in the elemental concentrations among substrates both before and after mushroom cultivation was evident. The studied elements accumulation in P. djamor was in an increasing trend in three subsequent flushes and was also reflected in the bioconcentration factors (BCFs). The highest BCF (2.35) was determined for Fe. Interestingly, the BCF values for all studied trace elements with detrimental health effects were lower than 1.00. The estimated daily intake (EDI) reflected that the P. djamor fruiting bodies grown on EMS can serve as an excellent dietary source of essential major and trace elements: Ca, Mg, Na, Mn, Mo, Ni, Se and Zn. On the other hand, EDI values for K, Cu, Fe, Ag, Ba, Cd, Al, Sb and Sr were greater than the referred guideline values corresponding to higher intake. Overall, the study presented an insight into elemental accumulations and demonstrated the potential utilisation of combined dairy manure-food waste digestate.