Aqueous biphasic systems containing non-ionic ethoxylated surfactants are perspective as media for some biochemical reactions. A comprehensive study of phase and aggregation behavior of these micellar systems, as well as consideration of the effects of biocomponents, their nature and concentration, pH and temperature are evident. In this work, the influence of salt medium (phosphate buffer), pH and temperature, as well as the presence of different bioorganic additives on cloud point and structural-thermodynamic properties (CMC and aggregation number) were studied for micellar solutions of non-ionic surfactants Triton X-114 and Tergitol NP-7 in water and buffers with pH 6.8, 4.3, 2.7. As additives were used the components of a biocatalytic hydrolysis reaction of penicillin G, namely penicillin G potassium salt, phenylacetic acid, and 6-aminopenicillanic acid. The different effects of the biocomponents on the cloud point and CMC value of Triton X-114 in the buffer with pH 4.3 are discussed. In addition, it was shown that the CMC value and the aggregation number of Triton X-114 decrease in buffer solutions (salting-out effect) and the CMC value increase at growing the temperature to 37 degrees C (what is typical for ethoxylated non-ionic surfactants). The addition of phenylacetic acid (10 mM) leads to a reduction in the aggregation number of Triton X-114 at pH 4.3; other bio-additives have practically no effect. Thus, the studied additives have different effects on the structure and phase behavior of investigated micellar solutions, and this is an important aspect for choosing an extraction system to carry out a bioreaction.