Abstract: To screen the safe and effective ingredients of coating agents for controlling tobacco rhizome diseases, the effects of four chemical fungicides (fludioxonil, tebuconazole, thifluzamide, difenoconazole), one plant growth regulator (brassinolide) and five biocontrol agents (Genkangzhuang, Heigenling, Miaoqiangzhuang, YX72, YX53) on tobacco seed germination and disease resistance were investigated by means of seed soaking and spore suspension root irrigation inoculation. The results showed that Genkangzhuang exhibited the best control efficacy against tobacco black shank and root rot caused by Fusarium oxysporum, with the relative control effects reaching 55.00% and 45.35%, respectively, which were higher than those of other treatments. However, this biocontrol agent showed no significant control effect on root rot caused by Fusarium solani, and exerted certain adverse effects on tobacco seed germination and seedling growth: the fresh and dry masss of above-ground parts were reduced by 21.96% and 0.72%, respectively, compared with the sterile water control. Brassinolide presented a significantly stronger growth-promoting effect than other agents, with the fresh and dry masss of above-ground parts and roots increased by 93.46%, 428.95%, 187.77% and 277.78%, respectively, while its control efficacy against tobacco black shank and F. oxysporum root rot was relatively low, only 16.67% and 11.63%, respectively, and it even posed a risk of exacerbating the severity of F. solani root rot. The combined seed soaking treatment with brassinolide and Genkangzhuang achieved a relative control effect of 54.38% against F. oxysporum root rot, which was higher than that of Genkangzhuang alone. No significant differences were observed in the disease severity of tobacco black shank and F. solani root rot between the combined treatment group and the control group. Moreover, the combined treatment significantly promoted the fresh mass accumulation of tobacco plants, with the fresh masss of above-ground parts and roots increased by 25.65% and 45.00%, respectively. In conclusion, the combined seed soaking treatment with Genkangzhuang and brassinolide integrated the disease control capacity of Genkangzhuang and the growth-promoting effect of brassinolide. It could effectively control F. oxysporum root rot while significantly promoting tobacco seedling growth, but its effects on tobacco black shank and F. solani root rot deserve further investigation.