Fusarium basal rot (FBR) of onion, which is caused by Fusarium oxysporum f.sp. cepae (Hanzawa) Snyder & Hansen (FOC) results in a substantial loss of marketable bulbs worldwide. One of the main reasons for the lack of FBR-resistant short-day cultivars is the unreliable screening methods available for the mature bulb stage when significant economic damage occurs. The objective of this study was to develop an artificial inoculation method with better quantification of inoculum for an effective selection of FBR-resistant mature onion bulbs. Mature bulbs of seven New Mexican short-day onion cultivars, along with susceptible and tolerant controls, were selected and evaluated for FBR resistance using mycelial and conidial inoculation methods, respectively. Transversely cut basal plates of mature bulbs were inoculated artificially with mycelia or conidia (12 × 105 spores/mL in 2014 and 3 × 105 spores/mL in 2015 embedded in potato dextrose agar plug) of a virulent FOC isolate ‘CSC-515’. Mature bulb evaluation using a visual rating scale (1 = no disease; 9 = >70% basal plate infected) revealed a high degree of FBR severity and incidence irrespective of the genetic background of the cultivars, minimizing the chance of disease escape, which is a significant problem in field inoculation. An attempt to inoculate intact basal plates postharvest resulted in minimal disease development, suggesting that mechanical resistance was conferred by the dry outer layer of the basal plate. The high selection pressure conferred by the conidial inoculation method developed in this study can effectively screen FBR-resistant onion bulbs to replace an unreliable field screening. Concentrations of the conidia lower than 3 × 105 spores/mL are recommended to detect subtle genetic differences in FBR resistance among the onion cultivars and their selected population.