Allelopathy tests were conducted on salt-tolerant transgenic eucalyptus trees conferring bacterial codA gene in the designated net-house conditions under Type II use (contained use) of the Japanese law on environmental biosafety aiming for Type I (field use) application. Three transgenic and corresponding nontransgenic genotypes were employed for four different tests: (1) sandwich bioassay; (2) soil germination method; (3) gas chromatography (GC) for volatile substances from the plants; and (4) high-performance liquid chromatography (HPLC) on phenolic compounds from fresh leaves, which are the primary allelopathic substances on the species. The simple approaches, the bioassays, indicated no significant difference between the transgenic and nongenetically modified genotypes. There was no qualitative difference between the transgenic and nontransgenic lines by GC or HPLC. Absence of any quantitative difference was suggested by repetitive examination and subsequent analysis of variance assessments with the chromatographic methods and bioassays. Moreover, it was also indicated that bioassays should be the primary assessment method for allelopathy in considering the simplicity, speed, low cost, and reproducibility of these methods. Overall, substantial equivalence was considered on the three transgenic genotypes with codA gene when compared with the nontransgenic Eucalyptus camaldulensis lines. The experiments supported the application to isolated field testing of the transgenic Eucalyptus camaldulensis genotypes as the first case and experience in Japanese regulatory approval processes Type I Use for the deliberate release to the environment.