ABSTRACT.- Fluorescent Pseudomonas have been extensively reported as effective biocontrol agents and can be used to manage Pythium seedling diseases in alfalfa. An in vivo methodology was developed to evaluate fluorescent Pseudomonas isolates for their ability to suppress disease and promote plant growth in the alfalfa-Pythium pathosystem. The standard method consists of a bioassay performed in growth chamber under controlled conditions of temperature and photoperiod. Alfalfa seeds were sown in commercial substrate and subsequently treated as follows:no Pseudomonas, no Pythium debaryanum (germination control); inoculated only with P. debaryanum (disease control); each one of the Pseudomonas isolates co-inoculated with P. debaryanum. An augmented experiment design was obtained by selecting a random complete blocks design for four checks systematically evaluated on 12 bioassays, and enlarging the blocks to accommodate up to 16 treatments per assay. Disease control recorded an emergence of 33.2% on day 15 after sowing date, as compared with germination control (100%). The 101 fluorescent Pseudomonas isolates showed a wide response on disease suppression ability. Twelve percent of tested isolates reasonably protected alfalfa plants against P. debaryanum, recording an emergence of over 60%. A similar procedure, in the absence of the pathogen, was used to evaluate alfalfa growth-promoting ability of selected Pseudomonas isolates. Five isolates exhibited a significant biocontrol activity against P. debaryanum and four of them also recorded a significant plant growth-promoting effect. Both bioassays were appropriate to identify candidate Pseudomonas isolates to be further tested under field conditions. Copyright (c) 2022 Agrociencia Uruguay