Charcoal rot and Fusarium wilt, caused by Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae, respectively, are major soil-borne diseases of strawberry that have caused significant crop losses in California. Anaerobic soil disinfestation has been studied as an industry-level option to replace soil fumigants to manage these serious diseases. Studies were conducted to discern whether Gramineae carbon input type, incubation temperature, or incubation duration influences the efficacy of this disease control tactic. In experiments conducted using ‘low rate’ amendment applications at moderate day/night temperatures (24/18 °C), and carbon inputs (orchard grass, wheat, and rice bran) induced an initial proliferation and subsequent decline in soil density of the Fusarium wilt pathogen. This trend coincided with the onset of anaerobic conditions and a corresponding generation of various anti-fungal compounds, including volatile organic acids, hydrocarbons, and sulfur compounds. Generation of these metabolites was associated with increases in populations of Clostridium spp. Overall, carbon input and incubation temperature, but not incubation duration, significantly influenced disease suppression. All Gramineae carbon inputs altered the soil microbiome and metabolome in a similar fashion, though the timing and maximum yield of specific metabolites varied with input type. Fusarium wilt and charcoal rot suppression were superior when anaerobic soil disinfestation was conducted using standard amendment rates of 20 t ha−1 at elevated temperatures combined with a 3-week incubation period. Findings indicate that anaerobic soil disinfestation can be further optimized by modulating carbon source and incubation temperature, allowing the maximum generation of antifungal toxic volatile compounds. Outcomes also indicate that carbon input and environmental variables may influence treatment efficacy in a target pathogen-dependent manner which will require pathogen-specific optimization of treatment protocols.