The functional relevance of microbial necromass in terrestrial biogeochemical cycles remains one of the unresolved mysteries of element cycling in ecosystems, especially considering the high microbial abundance and turnover in soil. We therefore established a protocol to manufacture multi-isotope (14C, 15N and 33P) labeled microbial necromass to comprehensively track the turnover of microbial necromass elements within element cycles. This protocol encompasses the i) microbial cultivation of Pseudomonas kilonensis ACN4 (Gram-negative) and Bacillus licheniformis DSM13 (Gram-positive) on labeled minimal medium as well as fungal cultivation of Hypsizygus tessulatus on a complex yeast medium, ii) quantification of radio- (14C, 33P) and stable (15N) isotope incorporation as well their cellular pool partitioning, and iii) determination of element and tracer isotope uptake efficiency. We achieved 1 g of bacterial biomass per liter minimum medium within 24 h and 2.9 g l−1 fungal biomass in complex medium within 18 d. This production rate enabled us to produce more than 100 g of necromass within only one half-life time of 33P, including post-harvest processing. Isotope uptake and incorporation for 33P ranged from 10 to 73%, for 15N from 24 to 52%, and for 14C from 12 to 23%. Each of the cultivated species showed individual patterns of tracer element uptake. The nutritional value of the carbon- (C), nitrogen- (N) and phosphorus- (P) labeled microbial necromass was characterized by a water-based, necromass species-specific partitioning scheme with subsequent elemental analysis of the pools. We separated Gram-negative, Gram-positive and fungi’s cellular pools to characterize element and tracer partitioning among dissolved versus particulate fractions. That is essential because these properties subsequently affect the respective pool's availability for ecosystem nutrition. Our procedure allows a defined production of microorganism-based necromass, enabling versatile use to determine necromass-related nutrient fluxes in terrestrial ecosystem studies.