SUMMARYPythium damping-off caused by Pythium aphanidermatum usually occurs in commercial nurseries of cucumber seedlings. The purpose of the present study was to develop a container medium for effectively suppressing Pythium damping-off of cucumber seedlings. Seven agricultural materials were tested for their suitability as substrates for the growth of cucumber seedlings. Bas Van Burren No. 4 peat moss (BVB) was able to promote the growth of cucumber seedlings, but it was ineffective against Pythium damping-off. Spent blewit mushroom compost (SBMC) was an available substrate that could inhibit P. aphanidermatum, but it also inhibited the growth of cucumber seedlings slightly. A cultural medium, spent blewit peat compost (SBPC), containing 50% (v/v) SBMC, 50% (v/v) BVB and 0·3% (w/v) lime was consequently formulated. The SBPC was able to promote the growth of cucumber seedlings and reduce the incidence of Pythium damping-off. A total of 20 micro-organisms were isolated from SBMC compost and screened in steamed SBPC medium for their ability to inhibit the growth of P. aphanidermatum. Among these, Bacillus aryabhattai isolate number CB13 was able to suppress P. aphanidermatum significantly in the steamed SBPC medium. Thus, bio-formulation of SBPC medium with the beneficial microbe B. aryabhattai CB13 was carried out. The incidence of Pythium damping-off was reduced from 58% in steamed SBPC medium to 4% in the bio-formulated container medium. The novel bio-formulation has high potential for controlling Pythium damping-off in commercial nurseries.